Optimization for speed and sensitivity in capillary high performance liquid chromatography. The importance of column diameter in online monitoring of serotonin by microdialysis

Abstract

The speed of a separation defines the best time resolution possible in online measurements using chromatography. The desired time resolution multiplied by the flow rate of the stream of analyte being sampled defines the maximum volume of sample per injection. The best concentration sensitivity in chromatography is obtained by injecting the largest volume of sample that is consistent with achieving a satisfactory separation, and thus measurement accuracy. Taking these facts together, it is easy to understand that separation speed and concentration sensitivity are linked in this type of measurement. To address the problem of how to achieve the best sensitivity and shortest measurement time simultaneously, we have combined recent approaches to the optimization of the separation itself with an analysis of method sensitivity. This analysis leads to the column diameter becoming an important parameter in the optimization process. We use these ideas in one particular problem presented by online microdialysis sampling/liquid chromatography/electrochemical detection for measuring concentrations of serotonin in the dialysate. In this case the problem becomes the optimization of conditions to yield maximum signal for a given sample volume under the highest speed conditions with a certain required number of theoretical plates. It turns out that the observed concentration sensitivity at an electrochemical detector can be regulated by temperature, particle size, injection volume/column diameter, and void time. The theory was successfully used for optimization of neurotransmitter serotonin measurement by capillary HPLC when sampling from a microdialysis flow stream. The final conditions are: 150 μm i.d., 3.1cm long columns with 1.7 μm particle diameter working at a flow rate of 12 μL/min, an injection volume of 500 nL, and a temperature of 343 K. The retention time for serotonin is 22.7s, the analysis time is about 36 s (which allows for determination of 3-methoxytyramine), and the sampling time is about 0.8 min with a perfusion flow rate of 0.6 μL/min.

Figure 1

The reduced plate height vs. reduced velocity (interstitial) generated from a column.

Figure 2

Apparent plate number measured at different injection volumes. Column: 100 μm inside diamter, 5.0 cm long capillary column packed with 1.7 μm BEH C 18 particles. Mobile phase: 100 mM sodium acetate, 0.15 mM disodium EDTA, 10.0 mM SOS, pH=4.0, mixed with 4% (v/v) acetonitrile. Flow rate: 4.0 μL/min. Column temperature T = 343 K.

Figure 3

Effect of particle size, injection volume and column temperature on concentration sensitivity and separation speed (void time t₀).

Figure 4

Concentration sensitivity as a function of void time t₀ and column diameter d_c with different particle sizes. Left: a line for each particle diameter in three dimensions. Right: projections of each of the latter curves in each of the two-dimensional planes.

Figure 5

Typical chromatogram of a 500 nL standard injection sample. Red: standard sample containing 2 nM 5-HT, 2 nM 3-MT and 7 μM AA. Black: blank aCSF solution. Column: 150-μ-i.d., 3.1-cm-length capillary column packed with BEH C 18 particles. Mobile phase: 100 mM sodium acetate, 0.15 mM disodium EDTA, 10.0 mM SOS, pH=4.0, mixed with 4% (v/v) acetonitrile. Flow rate: 12.0 μL/min. Column temperature: 70 °C. Electrochemical detection.

Figure 6

Determination of basal 5-HT and 3-MT in rat brain microdialysate. Separation conditions were the same as in Figure 5. Black: blank aCSF solution. Red: basal microdialysate sample collected from striatum of rat brain with a perfusion flow rate of 0.6 μL/min. Blue: 1:1 volume ratio mixture of basal microdialysate sample and 5 nM standard.

Figure 7

Monitoring of 5-HT concentration followed by high K⁺ stimulation. Administration: 120 mM K⁺ aCSF solution for 20 min. Samples were collected and analyzed offline. Separation and sampling conditions were the same as in Figure 6.

Figure 8

Chromatograms of samples with consecutive injections. Injection intervals: 0.6 minutes. Separation and sampling conditions were the same as in Figure 6.